CometAssay ES II. Instructions. Table of Contents. Catalog # ES. Catalog # ES. Electrophoresis system for the CometAssay

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1 Instructions For Research Use Only. Not For Use In Diagnostic Procedures CometAssay ES II Electrophoresis System for the CometAssay CometAssay ES II Catalog # ES Electrophoresis system for the CometAssay Table of Contents Page I. Introduction 1 II. Safety Information 2 III. Description of Equipment 3 IV. Materials Required But Not Supplied 3 V. Reagent Preparation 4 VI. Operation of Equipment 5 VII. Comet Assay Protocols 6 VIII. Data Analysis 8 IX. References 11 X. Related Products Available From Trevigen 11 XI. Appendices 13 XII. Troubleshooting Guide 15 Catalog # ES 2014 Trevigen, Inc. All rights reserved. Trevigen and CometAssay are registered trademarks, and CometSlide is a trademark of Trevigen, Inc

2 I. Introduction Trevigen s CometAssay provides a simple and effective method for evaluating DNA damage in cells. The principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of nucleoids under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when a current is applied. The sample is then visualized by epifluorescence microscopy. Evaluation of the DNA comet tail shape and migration pattern allows for assessment of DNA damage. The Neutral CometAssay is typically used to detect double-stranded breaks, whereas the Alkaline CometAssay is more sensitive, and is used to detect smaller amounts of damage including single and double-stranded breaks. In comet assays, cells are immobilized in a bed of low melting point agarose, on a Trevigen CometSlide. For both assay types, cells are lysed and the nucleoids subjected to electrophoresis. In the Alkaline CometAssay, samples are treated with alkali to unwind and denature the DNA and hydrolyze sites of damage prior to electrophoresis. Subsequent staining with a fluorescent DNA intercalating dye is performed for visualization. Quantitative and statistical data can readily be generated by analysis of the results using one of several commercially available image analysis software packages which calculate tail length, percent DNA in the tail, and tail moment. Since variability has been observed when performing the comet assay, Trevigen has developed a CometAssay Electrophoresis System (ES) II (Figure 1) optimized for use with Trevigen s CometAssay Kits (e.g. cat# K) and Control Cells (cat# CC and cat# NC). Electrophoresis may be performed using either Neutral Electrophoresis Buffer or Alkaline Electrophoresis Solution. Alkaline electrophoresis is more sensitive to variations in buffer height, temperature, and ion concentration, which affect DNA migration and can adversely impact results. Trevigen s CometAssay Electrophoresis System II, which can be ordered with, or without a separate power supply, overcomes assay variability first by placing an acrylic overlay on top of an elevated slide tray to maintain optimal buffer height for DNA migration. Secondly, a constant buffer temperature is maintained using an external chamber under the tank to cool the glass slide platform and buffer chamber. Electrophoresis time for optimal DNA migration is achieved in 30 minutes with Alkaline (ph>13) Electrophoresis Solution, or 45 minutes with Neutral Electrophoresis Buffer at 1V/cm. The smoke grey colored unit is designed to minimize exposure to UV light but still allow visual inspection. Third, specially designed slide trays are also provided to accommodate 2, 20 and 96 well slides and maintain proper slide orientation during electrophoresis. Trevigen s CometAssay Electrophoresis System, alkaline CometAssay Control Cells (cat# CC), and Neutral CometAssay Control Cells (cat# NC) enable investigators to consistently optimize their comet assays for highly reproducible results, and to standardize electrophoresis methods between individual users and laboratories. Figure 1: Trevigen s CometAssay Electrophoresis System II: II. Safety Information and Warnings 1. Caution! Electrical Hazard! This equipment is designed for use with a DC power supply providing up to 250 VDC. Careless handling can result in electrical shock. To avoid any risk of injury, the instrument should be operated in accordance with the instruction provided. Trevigen is not responsible for any injury or damage caused either by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not by Trevigen. Trevigen warrants to original purchaser a 1 year warranty for any defects in materials or workmanship under normal use. 2. Do not freeze unit. Never operate damaged equipment. 3. Do not cut cables. The CometAssay Electrophoresis System is designed with an Interlocking safety lid so no part of the active electrophoresis chamber is exposed during operation. Do not attempt to modify this safety design. 4. Always connect the cables to the power supply before turning the power supply on. 5. Never exceed maximum allowed voltage (250V) or current (450 ma). 6. Power to unit is to be supplied by an external DC-voltage power supply. 7. This instrument is designed and certified to meet IEC safety standards. 8. We recommend cleaning the unit with water and nonabrasive soap or detergent, followed by a rinse with deionized water. Avoid abrasive cleaners and rough cloths or brushes. Do not expose the apparatus to window sprays, phenol, acetone, benzene, halogenated hydrocarbon solvents, or undiluted laboratory alcohols. 9. Store at 4 C or room temperature. Do not expose the unit to prolonged exposure to UV light, or excessive heat such as dry heat sterilization or autoclaving. 1 2

3 III. Description of Equipment 1. Electrophoresis Tank with Leveling Bubble The electrophoresis tank is one continuous chemically resistant piece. Underneath the deck plate is an external chamber where the coolant is placed to absorb heat from the slide plate to help maintain a cool and even slide temperature during alkaline electrophoresis. The level of the electrophoresis system is adjusted using the four leveling feet with the leveling bubble resting on top of the deck plate. The recommended volume of electrophoresis buffer is 850 ml. Base dimensions are 21 cm x 29 cm. 2. Safety Lid and Cables The interlocking safety lid is made of black translucent smoke grey acrylic plastic with attached cables. The lid is designed to slide into a rectangular latch. The cables and latch are at opposite ends of the safety lid. Always check that the cables are properly connected to the electrodes. Please contact manufacturer if 4 mm cable adaptors are needed for BioRad power supply. 3. 2/20 (Cat# ES1) and 96 (Cat# ES2) Well Slide Trays The slide trays, machined from acrylic material, are designed to locate the slides in their running position and for easy slide removal at the completion of electrophoresis. The 2/20 Well Slide Tray holds ten 2-well or five 20-well slides. The 96 Well Slide Tray holds three 96-well slides. 4. Slide Tray Overlay The 1.3 cm acrylic Overlay is designed to sit on top of the Slide Tray and maintain a constant buffer height (current path) of 0.4 cm above the tray. The volume of electrophoresis buffer in the tank must be filled below the top of the overlay but above its base line for proper operation. IV. Materials Required But Not Supplied Equipment: 1. AC Power Supply designed to supply constant voltage, amperage, or power with automatic cross-over. Minimal Output specifications are 21V (constant) with fluctuating amperage to 400 ma. Note: Power supplies provided with orders: Cat# Item ESK-PS1 ES unit with power supply for North America ESK-PS2 ES unit with power supply for EU ESK-PS3 ES unit with power supply for UK ESK-PS4 ES unit with power supply for Switzerland ESK-PS5 ES unit with power supply for Australia µl, µl, 200 1,000 µl pipettors, and tips 3. Serological pipettor and pipettes 4. Boiling water bath and 37 C water bath 5. Epifluorescence microscope equipped with Fluorescein filter or light transmission microscope when using silver staining kit L graduated cylinder 7. 4 C refrigerator/cold room 3 Reagents: 1. Deionized water 2. 10X PBS, Ca ++ and Mg ++ free (cat# ) % Ethanol (reagent grade) 4. TE Buffer (10 mm Tris (ph 7.5), 1 mm EDTA) 5. CometAssay Kit (required: e.g. cat# K) ,000X SYBR Gold in DMSO For alkaline assays: 6. NaOH Pellets M EDTA (ph 8.0) 8. CometAssay Control Cells (cat# CC) 1 For neutral assays: 9. Tris Base 10. Ammonium Acetate 11. Sodium Acetate 12. Glacial Acetic Acid 13. Neutral CometAssay Control Cells (cat# NC) 1 Optional reagents: 14. Silver staining kit (cat# K) Dimethylsulfoxide 16. Tris Borate EDTA buffer 1 Available from Trevigen; refer to Section X for ordering information. V. Reagent Preparation Prepare one of the following electrophoresis solutions based on the sensitivity of assay desired. Additional reagents are required. For information regarding preparation of all needed reagents, please see the instructions for use for Trevigen s CometAssay (cat# K). For Alkaline CometAssay : 1. Alkaline Electrophoresis Solution ph>13 (200 mm NaOH, 1 mm EDTA) Prepare a stock solution of 500 mm EDTA, ph 8 (disodium salt) For 1 liter of electrophoresis solution: NaOH pellets 8 g 500 mm EDTA, ph 8 2 ml dh 2O to 1 liter (after NaOH is dissolved) Use of freshly made solution is recommended. Cool to 4 C. For Neutral CometAssay : 2. 1X Neutral Electrophoresis Buffer To prepare 10X Neutral Electrophoresis Buffer: Tris Base (mol. wt. = ) g Sodium Acetate trihydrate (mol. wt. = ) g 4

4 Dissolve in 450 ml of dh 2O. Adjust to ph = 9.0 with glacial acetic acid. Adjust volume to 500 ml, filter sterilize, and store at room temperature. Dilute the 10X stock to 1X in dh 2O to prepare 1 liter working strength buffer and cool tot 4 C. VI. Operation of Equipment A. Initial Setup 1. Slide coolant into the Coolant Chamber (Figure 1). Cool and store the unit in 4 C cold room or refrigerator. 2. Level electrophoresis tank. Level the electrophoresis tank using the leveling bubble provided by adjusting the four thumbscrew feet. B. Electrophoresis 1. Cool electrophoresis tank, tray, overlay and lid to 4 C by placing in cold room or refrigerator. Do not freeze the unit. Align the slots of the appropriate slide tray and place on glass slide platform. Inserting finger slots of slide tray adjacent to cathode (black) electrode is recommended. 2. Prepare Electrophoresis Solution (Buffer). Prepare 1L fresh as described in Reagent Preparation and cool to 4 C. Pour ~850 ml into cooled electrophoresis tank. 3. Perform Alkaline or Neutral Comet Quick Reference (VII). 4. Perform Electrophoresis. i. Immediately prior to electrophoresis, place electrophoresis unit at room temperature. ii. Insert slides into tray. Two well slides are locked into position by placing two slides into each position. Recommend always placing slide with Trevigen logo adjacent to cathode (black) electrode. DNA migrates towards the anode (red) during electrophoresis (Figure 1). iii. The 850 ml of Electrophoresis Solution (Buffer) should completely cover the slides. Carefully insert Slide Tray Overlay over slide tray by aligning slots. To ensure complete buffer coverage of slides, gradually lower Slide Tray Overlay in a manner similar to lowering a slide coverslip. iv. Verify that the Electrophoresis Solution (Buffer) is not above the Slide Tray Overlay. v. Set the power supply to 21 V (1 volt per cm). For Alkaline CometAssay apply voltage for 30 minutes for 2/20 well slides and 40 minutes for 96 well slides. For Neutral CometAssay apply voltage for 45 minutes for 2/20 well slides. Alkaline Tips: The Alkaline Electrophoresis Solution is a non-buffered system and temperature control is highly recommended. If the electrophoresis unit and buffer are cooled to 4 C, performance of electrophoresis at room temperature for minutes is acceptable. The cooled glass slide platform maintains the slide temperature. 5 In-house electrophoresis parameters with 200 mm NaOH/1mM EDTA Alkaline Solution, ph>13 at 4 C, has an amperage of ~220 ma. With increases in buffer temperature, the amperage increase is problematic for some power supplies with higher alkali concentrations. If the user prefers a higher alkali concentration, we recommend running at 0.7 volts per cm with a proportional increase in electrophoresis time. The Slide Tray Overlay is recommended for all Alkaline Electrophoresis. Any variation in buffer height directly affects DNA migration. Increase in buffer height results in slower DNA migration. The Slide Tray Overlay maintains a buffer height of 0.4 cm. Neutral Tips: Use of 1X Neutral Electrophoresis Buffer, based on in house experience, has amperage of ~110 ma when performed at 21V. The Neutral CometAssay will detect mainly double-stranded breaks. Neutral CometAssay images are different from Alkaline CometAssay images (VIII. Data Analysis, Figures 2 and 3). A high level of damage observed in Alkaline Comet does not necessarily mean damage will be observable using Neutral CometAssay and longer electrophoresis times (beyond 1 hour) may not necessarily improve results. VII. Comet Assay Protocols A. Alkaline Comet Quick Reference The Assay Protocol described below is written as a Quick Reference using alkaline Comet Control Cells (cat# CC) for two well slides. Reagents and detailed instructions including reagent preparation are provided with Trevigen s CometAssay Kits (See Section X). 1. Cool Lysis Solution at 4 C for at least 20 minutes before use. 2. Melt LMAgarose and cool in a 37 C water bath for at least 20 minutes. 3. Combine 50 µl of CCO (control cells) with 500 µl molten LMAgarose (at 37 C) and immediately pipette 50 µl onto two well CometSlide. Use side of pipette tip to spread agarose/cells over sample area. 4. Repeat step 3 for samples CC1, CC2, and CC3. 5. Place slides at 4 C in the dark for 10 minutes. 6. Immerse slides in 4 C Lysis Solution for minutes. 7. Immerse slides in 50 ml freshly prepared Alkaline Unwinding Solution, ph>13 for 20 minutes at room temperature, in the dark. 8. Perform Alkaline Electrophoresis as described in VI.B. 9. Immerse slides twice in dh 2O for 5 minutes each, then in 70% ethanol for 5 minutes. 10. Dry samples at 37 C for minutes. 6

5 11. Place 100 µl of diluted SYBR Gold onto each sample for 30 minutes. Remove excess SYBR solution and briefly rinse in water. Allow slide to dry completely at 37 C 12. View slides by epifluorescence microscopy. B. Neutral Comet Quick Reference For optimal results, the Neutral CometAssay should always be performed using Neutral CometAssay Control Cells (cat# NC). Please note that CometAssay Control Cells (cat# CC) are designed for Alkaline Comet assay only. 1. Cool Lysis Solution at 4 C for at least 20 minutes before use. 2. Melt LMAgarose and cool in a 37 C water bath for at least 20 minutes. 3. Combine 50 µl of NCO (control cells) with 500 µl molten LMAgarose (at 37 C) and immediately pipette 50 µl onto two well CometSlides. Use side of pipette tip to spread agarose/cells over sample area. 4. Repeat step 3 for samples NC1, NC2, and NC3, respectively. 5. Place slides at 4 C in the dark for 10 minutes. 6. Immerse slides in 4 C Lysis Solution for 1 hour. 7. Immerse slides in 50 ml of 4 C 1X Neutral Electrophoresis Buffer for 30 minutes. 8. Perform Neutral Electrophoresis as described in VI.B. 9. Immerse slides in DNA Precipitation Solution for 30 minutes at room temperature. 10. Immerse slides in 70% ethanol for 30 minutes at room temperature. 11. Dry samples at 37 C for minutes. 12. Place 100 µl of diluted SYBR Gold onto each sample for 30 minutes. Remove excess SYBR solution and briefly rinse in water. Allow slide to dry completely at 37 C. 13. View slides by epifluorescence microscopy. VIII. Data Analysis When excited ( nm) the DNA-bound SYBR Gold emits green light. In healthy cells the fluorescence is confined to the nucleoid (comprised of high molecular weight DNA): undamaged DNA is supercoiled and thus, does not migrate very far out of the nucleoid under the influence of an electric current. Whereas in cells that have accrued DNA damage, migrating fragments (comet tail) from the nucleoid (comet head) are observed. The negatively charged DNA migrates toward the anode and the extrusion length reflects increasing relaxation of supercoiling, which is indicative of damage. Common descriptors of DNA damage for alkaline comet assays are Percent DNA in the Tail, and Tail Moment. Percent DNA in the Tail is a normalized measure of the percent of total cell DNA found in the tail. Tail moment is a damage measure combining the amount of DNA in the tail with distance of migration. In neutral comet assays, Tail Moment is primarily used, since tail length continues to increase in contrast to alkaline comet tails which have finite lengths. Qualitative Analysis (Alkaline CometAssay ) The comet tail can be scored according to DNA content (intensity). The control (untreated cells) should be used to determine the characteristics of data for a healthy cell. Scoring can then be made according to nominal, medium or high intensity tail DNA content. At least 50 cells should be scored per sample. Quantitative Analysis (Alkaline and Neutral CometAssay ) There are several image analysis systems that are suitable for quantitation of CometAssay data. The more sophisticated systems include the microscope, camera and computer analysis package. These systems can be set up to measure the length of DNA migration, image length, nuclear size, and calculate DNA damage parameters. At least 50 randomly selected cells should be analyzed per sample. A list of commercially available software package is available from Trevigen. 7 8

6 Featured Data: Alkaline CometAssay In Figure 2a, data collected for each alkaline CometAssay Control Cell population (cat# CC) is shown as side-by side vertical box plots for comparison. The diamond shows the mean and confidence interval around the mean. The notched box shows the median, lower and upper quartiles, and the 75% confidence interval around the median. An example is provided below. Figure 2a: Box-Whisker plot of Control Cells: Percent DNA in Comet Tail % DNA in Tail Neutral CometAssay Data collected for each Neutral CometAssay Control Cell population (cat# NC) is provided below. Figure 3a: Box-Whisker plot of Neutral Control Cells: Tail Moment -10 CCO CC1 CC2 CC3 Control Cells % DNA by Etoposide n Mean SD SE 75% CI of Mean Median IQR 75% CI of Median CCO to to CC to to CC to to CC to to Figure 2b: Example comet tail shapes for each population. 9 Figure 3b: Example comet tail shapes for each population. 10

7 IX. References 1. Lemay, M. and K.A. Wood, Detection of DNA damage and identification of UVinduced photoproducts using the CometAssay kit. BioTechniques 27(4): Angelis, K.J., M. Dusinska and A.R. Collins Single cell gel electrophoresis: Detection of DNA damage at different levels of sensitivity. Electrophoresis 20: Morris, E.J., J.C. Dreixler, K-Y. Cheng, P.M. Wilson, R.M. Gin and H.M. Geller Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage. BioTechniques 26: Malyapa, R.S., C. Bi, E.W. Ahern, and J.L. Roti, Detection of DNA damage by the alkali comet assay after exposure to low dose gamma radiation. Radiation Res 149: Henderson, L., A. Wolfreys, J. Fedyk, C. Bourner, S. Windeback, The ability for the comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis 13: Visvardis, E.E., A.M. Tassiou, and S.M. Piperakis,1997. Study of DNA damage induction and repair capacity of fresh cryopreserved lymphocytes exposed to H 2O 2 and γ-irradiation with the alkaline comet assay. Mutation Res 383: Fairbairn, D.W., P.L. Olive, K.L. O'Neill The comet assay: a comprehensive review. Mutation Res 339: Collins, A.R., A.G. Ma, and S.J. Duthie, The kinetics of repair of oxidative DNA damage (strand breaks and oxidized pyrimidine dimers) in human cells. Mutation Res 336: Singh, N.P., M.T. McCoy, R.R. Tice, and E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: Östling, O. and K. J. Johanson, Microelectrophoretic study of radiation- induced DNA damage in individual cells. Biochem Biophys Res Commun 123: Singh, N.P., R.E. Stephens, Microgel electrophoresis: sensitivity, mechanisms, and DNA electrostretching. Mutation Res 383: Cosa, G, Focsaneanu, K. S, McLean, J.R.N., McNamee, J.P., Scaiano, J.C., Photophysical properties of fluorescent DNA-dyes bound to single- and doublestranded DNA in aqueous buffered solution. Photochemistry and Photobiology 73(6): X. Related Products Available From Trevigen Contact Trevigen for details of our unique product line for studying DNA damage and repair. All of Trevigen s kits include highly qualified enzymes, substrates, buffers, full instructions for use, and a synopsis specific for your kit. CometAssay Kits: Catalog # Description Size ESK CometAssay Starter Kit each K CometAssay 50 samples K CometAssay Silver Kit 50 samples K CometAssay Silver Staining Kit 200 samples K CometAssay Higher Throughput Kit 40 samples K CometAssay Kit 96 Wells 96 samples Control Cells: Catalog # Description Size CC CometAssay Control Cells (alkaline assay) 1 set NC Neutral CometAssay Control Cells 1 set 11 FLARE TM Assay Kit: Catalog # Description Damage Recognized Size FK FM Fpg Kit 8-oxoguanine, DNA containing formamidopyrimidine moieties 75 samples 100 samples FK FM hogg1 Kit 8-oxoguanine, DNA containing formamidopyrimidine moieties 75 samples 100 samples K-FK K-FM Endonuclease III Kit Thymine Glycol, 5,6-dihydrothymine, urea, 5-hydroxy-6- hydrothy-mine, 5,6-dihydrouracil, alloxan, 5-hydroxy-6- hydrouracil, uracil glycol, 5- hydroxy-5-methylhy-dantoin, 5-hydroxycytosine,5-hydroxyuracil, methyl-tartonylurea, thymine ring saturated or fragmentation product 75 samples 100 samples PARP Assay Kits: Catalog # Description Size K HT PARP in vivo Pharmacodynamic Assay II 96 tests K HT Universal Chemiluminescent PARP Assay 96 tests K HT w/histone Universal Coated Colorimetric Strip Wells PARP Assay 96 tests K HT w/histcoated Colorimetric Strip PARP/Apoptosis Wells Assay 96 tests K HT Chemiluminescent PARP/Apoptosis Assay 96 tests K HT Homogeneous PARP Inhibition Assay 96 tests Accessories: Catalog # Description Size FLARE TM Slides 100 slides SP FLARE TM Sample Prep >100 samples CometSlide (2 well) 25 slides CometAssay HT Slide (20 well) 10 slides Well CometSlide TM 10 slides K HT 8-oxo-dG ELISA Kit II 96 wells Fluorescence Mounting Medium 20 ml Hydrophobic Coverslips 100 each X PBS, Ca ++ and Mg ++ free 500 ml 12

8 XI. Appendices Appendix A Neutral CometAssay The CometAssay may be performed using neutral conditions that employ 1X TBE. Without treatment with Alkaline Buffer, this Neutral CometAssay will also detect mainly double-stranded breaks. 1. Cool Lysis Solution at 4 C for at least 20 minutes before use. 2. Melt LMAgarose and cool in a 37 C water bath for at least 20 minutes. 3. Combine 50 µl of cells at 1 x 10 5 /ml with 500 µl molten LMAgarose (at 37 C) and immediately pipette 50 µl onto two well CometSlides. Use side of pipette tip to spread agarose/cells over sample area. 4. Repeat step 3 for remaining samples. 5. Place slides at 4 C in the dark for 10 minutes. 5. Immerse slides in 4 C Lysis Solution for 30 minutes. 6. Immerse slides in 50 ml of 4 C 1X TBE buffer for 15 minutes. To prepare 10X TBE: Tris Base Boric Acid EDTA (disodium salt) 108 g 55 g 9.3 g Dissolve in 900 ml dh 2O. Adjust volume to 1 liter, filter sterilize, and store at room temperature. Dilute the 10X TBE to 1X in dh 2O to prepare 1 liter working strength buffer and cool to 4 C. 7. For the CometAssay ES II, add ~850 ml 4 C 1X TBE Buffer, place slides in electrophoresis slide tray and cover with Slide Tray Overlay. Set power supply to 21 volts and apply voltage for 40 minutes. 8. Immerse slides in dh 2O for 5 minutes. 9. Immerse slides in 70% ethanol for 5 minutes. 10. Dry samples at 37 C for minutes. 11. Place 100 µl of diluted SYBR Gold onto each sample and stain 30 minutes at room temperature in the dark. Remove excess SYBR solution. Allow slide to dry completely at 37 C. 12. View slides by epifluorescence microscopy. Appendix B Alternative Alkaline Electrophoresis Solution 300 mm NaOH, 1 mm EDTA ph>13: For 1 liter of electrophoresis solution: NaOH pellets 12 g 500 mm EDTA, ph 8 2 ml dh 2O (after NaOH is dissolved) add to: 1 liter Use of freshly made solution is recommended. Cool to 4 C. This amperage increases with 300 mm NaOH/1mM EDTA at 4 C and electrophoresis increased to minutes for 2 and 20 well slides. Appendix C DNA Stains 1. Important parameters to consider in choosing a DNA stain for the alkaline comet assay are similar fluorescence and decay rates for single- and doublestrand DNA. Table 1: DNA Stains Parameters (Cosa et al.) Dye Abs/Em ss:dsdna ss:dsdna Signal:Bkgrd (nm) fluorescence decay EtBr 520/ ~10 DAPI 356/ ~20 Propidium Iodide 536/ ~20 SYBR Gold 496/ >1000 SYBR Green 496/ >1000 YoYo-1 490/ ~ To use SYBR Green instead of SYBR Gold, simply prepare 1:10,000X SYBR Green I Staining Solution. The diluted stock is stable for several weeks when stored at 4 C in the dark. SYBR Green I (10,000X concentrate in DMSO) 1 µl TE Buffer, ph ml (TE: 10 mm Tris-HCl ph 7.5, 1 mm EDTA) 13 14

9 XII. Troubleshooting Guide General Problems PROBLEM CAUSE ACTION Cables do not fit BioRad powersupply Please contact manufacturer if 4 mm cable powersupply adaptors are needed for BioRad powersupply Unexpected and/or variety of tail shape. Cells in LMAgarose did not remain attached to the CometSlide. LMAgarose too hot Electrophoresis solution too hot. Cells were not washed to remove medium before combining with LMAgarose. Agarose percentage was too low. LMAgarose was not fully set before samples were processed. LMAgarose unevenly set on the slide. Rinsing steps too harsh. Specific to Alkaline Comet PROBLEM CAUSE ACTION Majority of cells in untreated control sample have large comet tails. Unwanted damage to cells occurred in culture or in sample preparations Majority of cells in untreated control sample have small to medium comet tails. In positive control (e.g. 100 µm hydrogen peroxide for 30 minutes on ice) no evidence of comet tail. Electrophoresis solution too hot Intracellular activity Endogenous oxidative damage or endonuclease activity after sample preparation is damaging DNA. No damage to DNA. Sample was not processed correctly. 15 Cool LMAgarose to 37 C before adding cells. Control temperature performing electrophoresis at 4 C. The ph of medium and carry over serum proteins, etc., can reduce the adherence of the agarose. Suspend cells in 1X PBS. Do not increase ratio of cells to molten agarose by more than 1 to 10. Ensure 0.5 mm dried ring due to agarose disc retraction is seen at the edge of the CometSlide TM area. Spread the agarose with the side of a pipette tip to ensure uniformity of agarose disc and better adherence. Gently place slides into solutions. Do not pour solutions over slides. Check morphology of cells to ensure healthy appearance. Handle cells or tissues gently to avoid physical damage. Control temperature by performing electrophoresis at 4 C. Keep cells on ice and prepare cell samples immediately before combining with molten LMAgarose. Ensure Lysis solution was chilled before use. Add DMSO to any cell sample that may contain heme groups. Ensure PBS used is calcium and magnesium free. Work under dimmed light conditions or under yellow light. Use fresh hydrogen peroxide to induce damage. Ensure each step in protocol was performed correctly. Failure to lyse, denature in alkali, or to properly perform electrophoresis may generate poor results. Specific to Alkaline Comet (continued) PROBLEM CAUSE ACTION Comet tails present but not significant in positive control. Please Recycle Insufficient denaturation in Alkaline Solution. Insufficient electrophoresis time. Specific to Neutral Comet 16 Increase time in Alkaline Solution up to 1 hour. Increase time of electrophoresis up to up to 1 hour for alkaline electrophoresis. Increase time of electrophoresis when running at cold temperatures. PROBLEM CAUSE ACTION In positive control no evidence of comet tail. Damaging agent doesn t cause double-strand breaks. Confirm damage by Alkaline Comet. Run Neutral Control Cells to confirm electrophoresis conditions. In positive control comet tails are extremely long and do not fit analysis window. Diffusion Artifacts are present; Difficulty distinguishing between head and tail Cells are necrotic or apoptotic. Electrophoresis time too long. Electrophoresis buffer conditions may not be optimal Increase treatment with damaging agent. Verify 75% viability. Decrease treatment with damaging agent. Decrease electrophoresis time to minutes. Use 1X Neutral Electrophoresis Buffer and follow protocol on page 7. The product accompanying this document is intended for research use only and is not intended for diagnostic purposes. Trevigen, Inc Helgerman Ct. Gaithersburg, MD Tel: Fax: info@trevigen.com